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1.
Gene Ther ; 31(3-4): 119-127, 2024 Mar.
Article in English | MEDLINE | ID: mdl-37833562

ABSTRACT

Dry gene powder is a novel non-viral gene-delivery system, which is inhalable with high gene expression. Previously, we showed that the transfection of p16INK4a or TP53 by dry gene powder resulted in growth inhibitions of lung cancer and malignant pleural mesothelioma (MPM) in vitro and in vivo. Here, we report that dry gene powder containing p53- expression-plasmid DNA enhanced the therapeutic effects of cisplatin (CDDP) against MPM even in the presence of endogenous p53. Furthermore, our results indicated that the safe transfection with a higher plasmid DNA (pDNA) concentration suppressed MPM growth independently of chemotherapeutic agents. To develop a new therapeutic alternative for MPM patients without safety concerns over "vector doses", our in vitro data provide basic understandings for dry gene powder.


Subject(s)
Lung Neoplasms , Mesothelioma, Malignant , Mesothelioma , Pleural Neoplasms , Humans , Mesothelioma/drug therapy , Mesothelioma/genetics , Powders/therapeutic use , Tumor Suppressor Protein p53/genetics , Pleural Neoplasms/drug therapy , Pleural Neoplasms/genetics , Cell Line, Tumor , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Cisplatin/pharmacology , Cisplatin/therapeutic use , Cisplatin/metabolism , DNA
2.
Pharmaceutics ; 15(12)2023 Dec 16.
Article in English | MEDLINE | ID: mdl-38140126

ABSTRACT

A number of functional nucleic acids, including plasmid DNA (pDNA) and small interfering RNA (siRNA), have been attracting increasing attention as new therapeutic modalities worldwide. Dry pDNA and siRNA powder formulations for inhalation are considered practical in clinical applications for respiratory diseases. However, physical stresses in the powder-forming process may destabilize nucleic acids, particularly when vectors with stabilizing effects are not used. We herein compare the stability of naked pDNA and siRNA through various physical treatments and two powder-forming processes. The structural and functional integrities of pDNA were markedly reduced via sonication, heating, and atomization, whereas those of siRNA were preserved throughout all of the physical treatments investigated. Spray-dried and spray-freeze-dried powders of siRNA maintained their structural and functional integrities, whereas those of pDNA did not. These results demonstrate that siRNA is more suitable for powder formation in the naked state than pDNA due to its higher stability under physical treatments. Furthermore, a spray-freeze-dried powder with a high content of naked siRNA (12% of the powder) was successfully produced that preserved its structural and functional integrities, achieving high aerosol performance with a fine particle fraction of approximately 40%.

3.
Sci Rep ; 12(1): 8634, 2022 05 23.
Article in English | MEDLINE | ID: mdl-35606391

ABSTRACT

Gene therapy using vectors has attracted attention in recent years for the treatment of cancers caused by gene mutations. Besides, new treatments are imperative for lung cancer, including non-small cell lung cancer (NSCLC) and malignant pleural mesothelioma (MPM), due to its high mortality. We developed a minimally invasive and orally inhalable tumor suppressor gene drug (SFD-p16 and SFD-p53) with non-viral vectors for lung cancer treatment by combining tumor suppressor genes with an inhalant powder that can deliver active ingredients directly to the lung. We used NSCLC (A549 and H1299) and MPM (H2052) cell lines in an air-liquid interface culture. Transfection of A549 and H2052 cells with SFD-p16 significantly increased p16 mRNA expression levels and decreased cell proliferation in both cell lines. Similar results were obtained with transfection of H1299 with the inhalable gene drug SFD-p53. In an in vivo experiment, a mouse model of lung cancer with orthotopically transplanted luciferase-expressing A549 cells was subjected to intratracheal insufflation of SFD-p16. Consequently, SFD-p16 effectively and directly affected lung cancer. This study suggests that inhalable gene drugs are effective treatments for NSCLC and MPM. We expect inhalable gene drugs to present a novel gene therapy agent for lung cancer that patients can self-administer.


Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Mesothelioma, Malignant , Mesothelioma , Pleural Neoplasms , Animals , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Non-Small-Cell Lung/therapy , Cell Line, Tumor , Genetic Therapy , Humans , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Lung Neoplasms/therapy , Mesothelioma/genetics , Mesothelioma/metabolism , Mesothelioma/therapy , Mice , Pleural Neoplasms/pathology , Transfection , Tumor Suppressor Protein p53/genetics
4.
Biol Pharm Bull ; 44(6): 822-829, 2021.
Article in English | MEDLINE | ID: mdl-34078814

ABSTRACT

It is well known that correct use of inhalers plays a critical role in optimal inhalation therapy, but the impact of incorrect inhaler use on pulmonary drug delivery has not been quantitatively evaluated. The aim of this study was to investigate the frequency of holding inhalers at incorrect angles during the drug-loading step while using Turbuhaler® and to quantify the influence of the inhaler angle on in vitro pulmonary delivery. Thirty patients prescribed Turbuhaler® at Shiga University of Medical Science Hospital were enrolled. During inhalation, the participants' inhalation techniques were assessed by clinical pharmacists. Additionally, the influence of the inhaler angle on pulmonary delivery of budesonide via Symbicort® Turbuhaler® was investigated using a Twin-Stage Liquid Impinger. Output efficiency (OE), stage 2 deposition (St2), and OE × St2 were calculated. An incorrect angle during the drug-loading step was observed in 33.3% of the participants. In vitro testing demonstrated that OE, an index of the loaded dose, significantly decreased by 73.3% at an incorrect angle, while St2, an index of the deagglomerating efficiency, was stable independent of the holding angle. OE × St2, indicating the bronchial and pulmonary drug delivery amount, decreased by 76.9%. An incorrect holding angle reduced the loaded dose, resulting in decreased pulmonary delivery. Error in the inhaler angle occurs frequently and demonstrates a considerable impact on pulmonary drug delivery. Hence, it is necessary to assess the Turbuhaler® angle during inhalation.


Subject(s)
Anti-Asthmatic Agents/administration & dosage , Budesonide, Formoterol Fumarate Drug Combination/administration & dosage , Dry Powder Inhalers , Medication Errors , Administration, Inhalation , Drug Delivery Systems , Humans
5.
Chem Pharm Bull (Tokyo) ; 68(7): 589-602, 2020.
Article in English | MEDLINE | ID: mdl-32611996

ABSTRACT

Inhaled lung cancer therapy is promising because of direct and noninvasive drug delivery to the lungs with low potential for severe systemic toxicity. Thus chemotherapeutic drugs have been administered clinically by nebulization of solution or suspension formulations, which demonstrated their limited pulmonary absorption and relatively mild systemic toxicity. In all these clinical trials, however, there was no obviously superior anticancer efficacy in lung cancer patients even at the maximum doses of drugs limited by pulmonary toxicity. Therefore methods that deliver both higher anticancer efficacy and lower pulmonary toxicity are strongly desired. In addition to the worldwide availability of pressured metered dose inhalers (pMDIs) and dry powder inhalers (DPIs) to treat local respiratory diseases, recent innovations in medicines and technologies are encouraging next steps toward effective inhaled lung cancer therapy with new therapeutic or drug delivery concepts. These include the discovery of target cells/molecules and drug candidates for novel cancer therapy, the development of high-performance inhalation devices for effective pulmonary drug delivery, and the establishment of manufacturing technologies for functional nanoparticles/microparticles. This review highlights the present situation and future progress of inhaled drugs for lung cancer therapy, including an overview of available inhalation devices, pharmacokinetics, and outcomes in clinical trials so far and some novel formulation strategies based on drug delivery systems to achieve enhanced anticancer efficacy and attenuated pulmonary toxicity.


Subject(s)
Antineoplastic Agents/therapeutic use , Drug Delivery Systems , Lung Neoplasms/drug therapy , Administration, Inhalation , Animals , Antineoplastic Agents/administration & dosage , Drug Compounding , Humans , Lung Neoplasms/pathology , Nebulizers and Vaporizers
6.
AAPS PharmSciTech ; 21(2): 53, 2020 Jan 06.
Article in English | MEDLINE | ID: mdl-31907655

ABSTRACT

To achieve adequate inhalation therapy, a proper inhalation technique is needed in clinical practice. However, there is limited information on proper inhalation flow patterns of commercial inhalers. Here, we quantitatively estimated airway deposition of two commercial pressurized metered dose inhalers (pMDIs) to determine their optimal inhalation patterns. Sultanol® inhaler (drug particles suspended in a propellant, suspension-pMDI) and QVAR™ (drug dissolved in a propellant with ethanol, solution-pMDI) were used as model pMDIs. Aerodynamic properties of the two pMDIs were determined using an Andersen cascade impactor with human inhalation flow simulator developed by our laboratory. As indices of peripheral-airway drug deposition, fine particle fractions (FPFPA) at different inhalation flow rates were calculated. The time-dependent particle diameters of sprayed drug particles were determined by laser diffraction. On aerodynamic testing, FPFPA of suspension-pMDI significantly decreased depending on the increasing inhalation flow rate, while solution-pMDI achieved higher and constant FPFPA in the range of the tested inhalation flow rates. The particle diameter of solution-pMDI markedly decreased from 5 to 3 µm in a time-dependent manner. Conversely, that of suspension-pMDI remained at 4 µm during the spraying time. Although "slow inhalation" is recommended for pMDIs, airway drug deposition via solution-pMDI (extra-fine particles) is independent of patients' inhalation flow pattern. Clinical studies should be performed to validate instruction for use of pMDIs for each inhaler for the optimization of inhalation therapy.


Subject(s)
Albuterol/administration & dosage , Metered Dose Inhalers , Administration, Inhalation , Humans , Particle Size
7.
Int J Pharm ; 574: 118880, 2020 Jan 25.
Article in English | MEDLINE | ID: mdl-31811928

ABSTRACT

We evaluated the storage stability of powder containing naked plasmid DNA (pDNA) and hyaluronic acid (HA) or mannitol (Man) prepared by the spray-freeze-drying technique, through which we have reported high gene expression without any gene vectors. The powders composed of 5-10-µm porous particles and showing excellent dispersion were stored for 12 months under three storage conditions: 5 °C/Dry, 25 °C/Dry, and 25 °C/75% relative humidity. The humidified powders lost their porous shape within 1 week and were not suitable for inhalation characterization. On the other hand, the powders under dry conditions maintained high inhalation characteristics and pDNA integrity for 12 months. We administered the powders to the lungs of mice. The naked pDNA in HA powder showed significantly higher gene expression compared with that in Man powder and a pDNA-polyethylenimine complex solution. The gene expression of pDNA/HA powder was maintained for 12 months. These results suggest that powder containing naked pDNA is stable on storing under appropriate dry conditions and the naked pDNA/HA powder shows effective pulmonary gene expression.


Subject(s)
DNA/genetics , Gene Expression/genetics , Hyaluronic Acid/chemistry , Lung/drug effects , Powders/chemistry , Powders/pharmacology , Administration, Inhalation , Animals , Dry Powder Inhalers/methods , Female , Genetic Vectors/genetics , Mannitol/chemistry , Mice , Mice, Inbred ICR , Particle Size , Plasmids/genetics , Polyethyleneimine/chemistry
8.
Int J Pharm ; 565: 294-305, 2019 Jun 30.
Article in English | MEDLINE | ID: mdl-31078647

ABSTRACT

We performed in vivo/ex vivo/polyacrylamide gel electrophoresis (PAGE) fluorescence imaging of near-infrared fluorescence (NIRF)-labeled siRNA (Cy5.5-siGL3) in mice to investigate the validity of each fluorescence imaging result as the biodistribution/biostability assessment of siRNA. Statistically significant correlations could be obtained between the in vivo and ex vivo fluorescence intensities of Cy5.5 in the relevant regions/tissues, except the lung region/tissue after intravenous administration. On PAGE fluorescence images with the naked formulation, there was no band corresponding to intact Cy5.5-siGL3 from all the tissues evaluated after intravenous administration, indicating that the fluorescence detected by in vivo and ex vivo fluorescence imaging was derived from degraded Cy5.5-siGL3 or free Cy5.5 cleaved from Cy5.5-siGL3. However, the band was detected from the lungs after intratracheal administration of the naked formulation, confirming higher stability of siRNA on the respiratory epithelium than in the blood. Regarding the polyethyleneimine formulation, the band was detected from all the tissues evaluated after intravenous administration and from the lungs after intratracheal administration, verifying the enhanced stability of siRNA in the body. These results clearly indicated the necessity of comprehensive analysis from in vivo/ex vivo/PAGE fluorescence imaging to precisely assess the distribution and stability of NIRF-labeled oligonucleotides including siRNA in the body.


Subject(s)
Carbocyanines/administration & dosage , Polyethyleneimine/administration & dosage , RNA, Small Interfering/administration & dosage , Administration, Inhalation , Administration, Intravenous , Animals , Carbocyanines/pharmacokinetics , Cell Line, Tumor , Electrophoresis, Polyacrylamide Gel , Female , Humans , Luciferases, Firefly/genetics , Lung/metabolism , Mice, Inbred ICR , Optical Imaging , Polyethyleneimine/pharmacokinetics , RNA, Small Interfering/pharmacokinetics , Tissue Distribution
9.
J Pharm Sci ; 108(8): 2661-2667, 2019 08.
Article in English | MEDLINE | ID: mdl-30954524

ABSTRACT

In order to evaluate the in vivo effect of inhaled formulations, it is a gold standard to create a lung metastasis model by intravenously injecting cancer cells into an animal. Because the cancer grows from the blood vessel side, there is a possibility of underestimating the effect of an inhaled formulation administered to the lung epithelium side. In addition, the metastasis model has disadvantages in terms of preparation time and expense. The present study aimed to establish a new method to evaluate the effect of an inhaled small interfering RNA (siRNA) formulation that is more correct, more rapid, and less expensive. We investigated whether siRNA can suppress gene expression of plasmid DNA (pDNA) by serial pulmonary administration of siRNA and pDNA powders prepared by spray-freeze-drying. We revealed that formulations of dry siRNA powder significantly suppressed gene expression of pDNA powder compared with a control group with no siRNA. Naked siRNA inhalation powder with no vector showed the suppression of gene expression equivalent to that of an siRNA-polyethyleneimine complex without damaging tissues. These results show that the present method is suitable for evaluating the gene-silencing effect of inhaled siRNA powders.


Subject(s)
DNA/administration & dosage , Gene Silencing , Plasmids/administration & dosage , RNA, Small Interfering/administration & dosage , Administration, Inhalation , Animals , DNA/genetics , Female , Freeze Drying , Lung/metabolism , Mice , Mice, Inbred ICR , Plasmids/genetics , Powders , RNA, Small Interfering/genetics
10.
J Vet Med Sci ; 81(4): 577-585, 2019 Apr 16.
Article in English | MEDLINE | ID: mdl-30828031

ABSTRACT

Bovine viral diarrhea (BVD) is a chronic disease of cattle caused by infection with BVD virus (BVDV) and can result in economic losses within the livestock industry. In Japan, the test and culling policy is a basic control measure, and implementation of an adequate vaccination program is recommended as a national policy. In addition, optional control measures, including compulsory testing of introduced animals and bulk tank milk (BTM) testing as a mass screening method, are used in several provinces, but their efficacy has not been completely assessed. We evaluated these control measures using the scenario tree model of BVD in Japan, developed in the previous study. The model outputs indicated that compulsory testing of all introduced cattle, rather than only heifers and/or non-vaccinated cattle, was cost effective and reduced the risk of BVDV introduction due to animal movement and that BTM testing could effectively monitor most part of the cattle population. Vaccination coverage and BVDV prevalence among introduced cattle could also affect the cost effectiveness of compulsory testing of targeted cattle, particularly under low vaccination coverage or high BVDV prevalence. However, even with the implementation of a highly effective monitoring scheme for many years, BVD risk could not be eliminated; it instead converged at a very low level (0.02%). Disease models with a cost-effective output could be a powerful tool in developing a control scheme for chronic animal diseases, including BVD, with the consent of relevant stakeholders.


Subject(s)
Bovine Virus Diarrhea-Mucosal Disease/prevention & control , Cost-Benefit Analysis , Diarrhea Viruses, Bovine Viral/isolation & purification , Milk/virology , Animals , Bovine Virus Diarrhea-Mucosal Disease/economics , Bovine Virus Diarrhea-Mucosal Disease/epidemiology , Cattle , Female , Japan/epidemiology , Models, Statistical , Prevalence , Vaccination/veterinary , Viral Vaccines/therapeutic use
11.
Mol Pharm ; 16(2): 489-497, 2019 02 04.
Article in English | MEDLINE | ID: mdl-30092131

ABSTRACT

Gene therapy is a breakthrough treatment strategy against several intractable and lethal diseases that previously lacked established treatments. Viral and nonviral vectors have been studied to realize higher gene transfection efficiencies and to suppress the degradation of gene by nucleolytic enzymes in vivo. However, it is often the case that the addition of a vector results in adverse effects. In this study, we identified formulations of dry naked plasmid DNA (pDNA) powders with no vector showing significantly higher gene expression than pDNA solutions including vectors such as polyethylenimine (PEI) in the lungs of mice. We prepared the naked pDNA powders by spray-freeze-drying with various excipients. The gene expression of naked pDNA powders exceeded those of pDNA solutions containing PEI, naked pDNA solution, and reconstituted pDNA powder. Gene expression of each naked pDNA powder was dependent on the composition of excipients. Among them, the mice that were administered the pDNA powder composed of low-molecular-weight hyaluronic acid (LHA) as an excipient showed the highest gene expression. The lactate dehydrogenase activity and concentration of inflammatory cytokines in bronchoalveolar lavage fluid were comparable to those caused by ultrapure water. The results suggest that useful dry naked nucleic acid powders for inhalation could be created by optimizing the excipients, offering new insights into the development of pulmonary gene therapy.


Subject(s)
Genetic Therapy/methods , Hyaluronic Acid/administration & dosage , Hyaluronic Acid/chemistry , Lung/metabolism , Plasmids/genetics , Plasmids/metabolism , Administration, Inhalation , Animals , Female , Genetic Vectors/chemistry , Mice , Molecular Weight , Polyethyleneimine/chemistry , Powders/administration & dosage , Powders/chemistry
12.
Mol Ther Nucleic Acids ; 12: 698-706, 2018 Sep 07.
Article in English | MEDLINE | ID: mdl-30092405

ABSTRACT

Inhalation therapy using small-interfering RNA (siRNA) is a potentially effective therapeutic strategy for lung cancer because of its high gene-silencing effects and sequence specificity. Previous studies reported that intratracheal administration of siRNA using pressurized metered dose inhalers or nebulizers could suppress tumor growth in murine lung metastatic models. Although dry powder inhalers are promising devices due to their low cost, good portability, and preservability, the anti-tumor effects of siRNA dry powder have not been elucidated. To evaluate the gene-silencing and anti-tumor effects of intratracheally delivered siRNA dry powder, vascular endothelial growth factor-specific siRNA (VEGF-siRNA) dry powder was administered intratracheally to mice with metastatic lung tumors consisting of B16F10 melanoma cells or Lewis lung carcinoma cells. A single intratracheal administration of VEGF-siRNA dry powder reduced VEGF levels in both bronchoalveolar lavage fluid and lung tumor tissue. Furthermore, repeated intratracheal administration of VEGF-siRNA dry powder suppressed the number of visible metastatic foci on the lung surface and tumor area in lung tissues. Taken together, intratracheal administration of siRNA dry powder could be a novel therapeutic strategy for lung cancer through the suppression of specific genes expressed in lung tumor tissue.

13.
Biol Pharm Bull ; 41(6): 937-943, 2018.
Article in English | MEDLINE | ID: mdl-29863082

ABSTRACT

In the development of drugs for intra-articular administration, sustained-release formulations are desirable because it is difficult to maintain the effect of conventional injections due to immediate drug leakage from the joint cavity. In this study, a sustained-release poly(lactic-co-glycolic acid) (PLGA) microsphere formulation for intra-articular administration containing indocyanine green (ICG) as a model drug was prepared to follow its fate after intra-articular administration in rats with a real-time in-vivo imaging system. ICG administered as an aqueous solution leaked from the joint cavity in a short time and was excreted outside the body within 1-3 d. However, ICG in the sustained-release formulation was retained in the joint cavity and released for 2 weeks. Next, a sustained-release formulation containing PLGA microspheres in a hyaluronic acid (HA) gel formulation was prepared. After gradual release in two stages, we could achieve sustained release for a longer period. It is considered that a combination formulation of PLGA microspheres and HA gel can significantly improve the sustained release of a drug administered into the knee joint.


Subject(s)
Hyaluronic Acid/administration & dosage , Lactic Acid/administration & dosage , Polyglycolic Acid/administration & dosage , Animals , Delayed-Action Preparations/administration & dosage , Delayed-Action Preparations/chemistry , Drug Administration Schedule , Drug Compounding , Drug Liberation , Gels , Hyaluronic Acid/chemistry , Injections, Intra-Articular , Knee Joint , Lactic Acid/chemistry , Male , Microspheres , Polyglycolic Acid/chemistry , Polylactic Acid-Polyglycolic Acid Copolymer , Rats, Sprague-Dawley
14.
J Control Release ; 279: 99-113, 2018 06 10.
Article in English | MEDLINE | ID: mdl-29627404

ABSTRACT

In the present study, a novel dry small interfering RNA (siRNA) powder for inhalation, containing polyethyleneimine (PEI) as a delivery vector, was produced by spray freeze drying (SFD). The powder had spherical and highly porous structure of approximately 10 µm in diameter with high aerosol performance for emission and lung delivery. The reconstituted siRNA/PEI complex after dissolution of the powder had almost the same physicochemical properties and in vitro gene silencing activity as the original one constituted in the sample solution before SFD, showing that the integrity of the siRNA was maintained. In in vivo studies of intratracheal administration into lung metastasis mice and healthy mice, powder with a low dose of 3 µg siRNA exhibited strong and specific gene silencing activity against tumors metastasized to the lungs, whereas it caused no significant histological changes, lactate dehydrogenase leakage, or inflammatory cytokine induction in the lungs. These results strongly indicated that inhalable dry siRNA/PEI powders can provide effective pulmonary gene silencing without severe lung injury and that SFD can be applied to the production of such powders.


Subject(s)
Gene Silencing , Lung Neoplasms/therapy , Polyethyleneimine/chemistry , RNA, Small Interfering/administration & dosage , Administration, Inhalation , Aerosols , Animals , Dry Powder Inhalers , Female , Freeze Drying , Lung/metabolism , Lung/pathology , Lung Neoplasms/genetics , Lung Neoplasms/secondary , Male , Mice , Mice, Inbred BALB C , Mice, Inbred ICR , Particle Size , Porosity , Powders
15.
J Pharm Sci ; 107(6): 1731-1735, 2018 06.
Article in English | MEDLINE | ID: mdl-29428382

ABSTRACT

This study aimed at developing a novel analytical method to identify optimal inhalation flow patterns for commercial dry powder inhalers (DPIs) and pressurized metered dose inhalers (pMDIs). As typical commercial DPI and pMDI, Pulmicort® Turbuhaler®, and Sultanol® Inhaler were evaluated by an in vitro inhalation performance testing system with a flow pattern simulator. An 8-stage Andersen cascade impactor (ACI) or twin stage liquid impinger (TSLI) was applied to determine the inhalation performance. The peak flow rate (PFR) of the inhalation flow pattern was set from 15 to 80 L/min in reference to our previous study. From TSLI test results, a higher PFR improved the inhalation performance of the DPI, while the performance of the pMDI was less affected by the PFR. Conversely, from ACI test results, the pMDI performance decreased with a higher PFR, while the DPI followed a similar pattern as in the TSLI test results, because ACI is a finer aerodynamic classification apparatus than TSLI. These results suggested that our in vitro system using a human inhalation flow pattern simulator successfully detected different optimal inhalation patterns between DPI and pMDI. That is, the higher PFR is better for Pulmicort® Turbuhaler® (DPI). Conversely, lower PFR is desirable for Sultanol® Inhaler (pMDI).


Subject(s)
Albuterol/administration & dosage , Bronchodilator Agents/administration & dosage , Budesonide/administration & dosage , Dry Powder Inhalers , Metered Dose Inhalers , Administration, Inhalation , Equipment Design , Humans , Particle Size
17.
J Vet Med Sci ; 79(7): 1172-1181, 2017 Jul 07.
Article in English | MEDLINE | ID: mdl-28539533

ABSTRACT

A scenario tree model was developed to propose efficient bovine viral diarrhea (BVD) control measures. The model used field data in eastern Hokkaido where the risk of BVDV infection in cattle has been reduced by an eradication program including mass vaccination, individual tests prior to communal pasture grazing, herd screening tests using bulk milk, and outbreak investigations of newly infected herds. These four activities were then used as hypothesized control measures in the simulation. In each simulation, the numbers of cattle infected persistently and transiently with BVDV detected by clinical manifestations and diagnosis tests and of missed by all of the diagnosis tests were calculated, and the numbers were used as indicators to be compared for the efficacy of the control measures. The model outputs indicated that the adoption of mass vaccination decreased the number of missed BVD cattle, although it did not increase the number of detected BVD cattle. Under implementation of mass vaccination, the efficacy of individual tests on selected 20% of the young and adult cattle was equal to that of the herd screening test performed in all the herds. When the virus prevalence or the number of sensitive animals becomes low, the efficacy of herd screening test was superior to one of individual tests. Considering the model outputs together, the scenario tree model developed in the present study was useful to compare the efficacy of the control measures for BVD.


Subject(s)
Bovine Virus Diarrhea-Mucosal Disease/prevention & control , Diarrhea Viruses, Bovine Viral , Animals , Bovine Virus Diarrhea-Mucosal Disease/epidemiology , Cattle , Disease Outbreaks/prevention & control , Disease Outbreaks/veterinary , Female , Japan/epidemiology , Models, Statistical , Program Evaluation , Viral Vaccines/therapeutic use
18.
Biol Pharm Bull ; 40(6): 830-836, 2017 Jun 01.
Article in English | MEDLINE | ID: mdl-28100866

ABSTRACT

In the development of a drug for intra-articular administration, a sustained-release formulation is desirable since it is difficult to sustain the effects of conventional injections due to fast drug leakage from the joint cavity. In this study, we prepared sustained release gel formulations for intra-articular administration containing indocyanine green (ICG) as a model drug to follow its fate after intra-articular administration in rats with in-vivo imaging system (IVIS). ICG administered as an aqueous solution leaked from the joint cavity in a short time and was excreted out of the body within a day. On the other hand, ICG in the sustained-release formulations was retained and released in the joint cavity for a week. Next, we prepared a sustained-release formulation with hyaluronic acid (HA) as the gel base containing a pain-relief drug (Drug A). We had administered it and other formulations into the rat knee where we injected bradykinin to evaluate their walking distance after 1 and 3 d. The effect of an aqueous solution of Drug A disappeared on day 3. The HA gel formulation without Drug A was more effective than the aqueous solution. The HA gel formulation with Drug A was the most effective; the walking distance was about 85% of the baseline on day 3. This study showed that the gel formulations were effective to sustain the release of a drug in the knee joint, and that the combination of a pain-relief drug with HA gel was effective to improve the mobility of the acute pain model rats.


Subject(s)
Analgesics/administration & dosage , Hyaluronic Acid/administration & dosage , Knee Joint/metabolism , Pain/drug therapy , Analgesics/pharmacology , Animals , Bradykinin , Coloring Agents/administration & dosage , Delayed-Action Preparations/administration & dosage , Delayed-Action Preparations/pharmacology , Gels , Hyaluronic Acid/pharmacology , Indocyanine Green/administration & dosage , Injections, Intra-Articular , Male , Pain/chemically induced , Rats, Sprague-Dawley , Walking
19.
Biol Pharm Bull ; 39(3): 394-400, 2016.
Article in English | MEDLINE | ID: mdl-26725531

ABSTRACT

Cationic polymers are being studied as non-viral gene delivery vectors. Poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (PAsp(DET)) and their block copolymers with poly(ethylene glycol), PEG-PAsp(DET), have been reported as efficient biodegradable non-viral vectors which form a polyplex with plasmid DNA (pDNA). However, the polyplexes are not stable because PAsp(DET) and PEG-PAsp(DET) are easily subjected to hydrolysis; therefore, they need to be prepared on site. In this study, using the biodegradable polycations as non-viral vectors, PAsp(DET) and PEG-PAsp(DET), we investigated the effects of L-leucine (Leu) on the polyplex. We prepared solutions and dry powders with and without Leu. Both dry powders had large and porous particles and Leu acted as a dispersing agent. The transfection activity of the sample solutions decreased within a month. However, the decrease in the transfection activity was partially suppressed by the dry powder with Leu at 5 and 25°C at 3 months. Furthermore, transfection experiments revealed that Leu exhibited a pDNA-stabilizing effect in the solution and dry powder. Similar results were observed for pDNA integrity, where a polyplex was formed in the dry powder. The results suggest that Leu is a candidate stabilizer to protect pDNA from degradation.


Subject(s)
DNA/chemistry , Leucine/chemistry , Peptides/chemistry , Polyethylene Glycols/chemistry , Administration, Inhalation , Animals , Cell Line, Tumor , DNA/administration & dosage , Drug Compounding , Drug Stability , Gene Expression , Luciferases, Firefly/genetics , Mice , Particle Size , Plasmids , Powders
20.
Biol Pharm Bull ; 39(3): 368-77, 2016.
Article in English | MEDLINE | ID: mdl-26754254

ABSTRACT

Most safety evaluations of dry powder inhalers (DPIs) using cultured cells have been performed with dry powder formulations dissolved in a medium. However, this method is not considered to be suitable to evaluate the safety of inhaled dry powder formulations correctly since it cannot reflect the actual phenomenon on the respiratory epithelial surface. In this study, we established a novel in-vitro safety evaluation system suitable for DPIs by combining an air-liquid interface cultured cell layer and a device for dispersing dry powders, and evaluated the safety of candidate excipients of dry powders for inhalation. The safety of excipients (sugars, amino acids, cyclodextrins, and positive controls) in solutions was compared using submerged cell culture systems with a conventional 96-well plate and Transwell(®). The sensitivity of the cells grown in Transwell(®) was lower than that of those grown in the 96-well plate. Dry powders were prepared by spray-drying and we evaluated their safety with a novel in-vitro safety evaluation system using an air-liquid interface cultured cell layer. Dry powders decreased the cell viability with doses more than solutions. On the other hand, dissolving the dry powders attenuated their cytotoxicity. This suggested that the novel in-vitro safety evaluation system would be suitable to evaluate the safety of DPIs with high sensitivity.


Subject(s)
Cell Culture Techniques , Dry Powder Inhalers , Excipients/pharmacology , Air , Cell Line, Tumor , Cell Survival/drug effects , Chemistry, Pharmaceutical , Humans , Particle Size , Powders
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